Human CD20 is highly expressed in B-lymphoma cells and B-lymphocytes. Once expressed, CD20 remains on cell surface without being internalized or secreted. Moreover, antibody (Abs) binding to CD20 trigger apoptotic cell signaling resulting in the cell death. These attributes make CD20 an ideal therapeutic target for B-lymphomas. Many monoclonal antibodies specifically binding to extracellular large loop formed by residues 142-187 of human CD20 are developed and commonly used for treating non-Hodgkin's lymphoma. The US Food and Drug Administration approved a monoclonal antibody, Rituximab (RTX), a human/mouse chimeric anti-CD20 antibody, for the treatment of B-cell Non-Hodgkin lymphomas (NHLs) in 1997. RTX has been widely used in treating patients with B-cell lymphomas, but only 10% of the treated patients have complete responses. RTX has also for the treatment of auto-immune diseases, such as rheumatoid arthritis. Thus, there is still a need to develop alternative CD20 specific Ab that may provide better therapeutic results or be useful for diagnostic applications.
Single domain antibodies (sdAb, also called nanobodies) are small antigen binding domains (˜15 kD) with better penetration in tissues than intact conventional antibodies. Nanobodies are encoded by variable regions of heavy chain variable region (VHH) DNA derived from heavy chain antibodies (HcAb) exclusive to Camelids. Although sdAbs have been evaluated for therapeutic and imaging applications, sdAb specific to human CD20 are not believed to have been previously been described. Most current CD20 immuno-reagents are mAbs and their scFv derivatives. Due to their smaller size, sdAbs have improved tumor penetration compared to full length conventional antibodies. An additional consequence of this property is their rapid normal tissue clearance, making them compatible for use with short half-life radionuclides such as 68Ga and 18F for PET imaging and 211At for targeted radiotherapy (Vaneycken et al., 2011; Pruszynski et al., 2012). SdAbs are able to direct treatment to the tumors with minimal off target impact, which should greatly enhance the prospects for patients.